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We have studied gene transfer efficiency of glycosylated polylysines and glycosylated polyethylenimines as vectors in immortalized differentiated airway gland serous cells and primary cultures of human airway surface epithelial cells. In both cell types, lactosylated PEI was more efficient for gene transfer than unsubstituted PEI and lactosylated polylysine which requires the presence of endosomolytic agents. However, for all the vectors tested, gene transfer efficiency was lower in differentiated cells as compared with poorly differentiated cells. The presence of membrane lectins, i.e. cell surface sugar-specific receptors, was evaluated using fluorescein-conjugated neoglycoproteins and microscopy or flow cytometry. In differentiated airway surface epithelial cells, membrane lectins were not expressed and plasmid DNA/fluorescein-conjugated glycosylated polymer complexes were not incorporated. This accounted in part for the lack of gene transfer efficiency in these cells. In contrast, in differentiated airway gland serous cells, expression of lectins and their endocytotic properties appeared to be similar to that observed in undifferentiated cells, and plasmid DNA/fluorescein-conjugated glycosylated polymer complexes were incorporated in similar amounts by cells in both differentiated states. Glycosylated PEI appears to be a promising gene delivery system since it is more efficient than the sugar-free polymer and does not require endosomolytic agents. However, in differentiated airway gland serous cells, a low gene transfer efficiency was observed that could not be attributed to low expression of membrane lectins or low uptake of glycosylated complexes. An impaired intracellular trafficking of glycosylated complexes in differentiated airway gland serous cells is suggested. Copyright © 2002 John Wiley & Sons, Ltd.

Knowing that human blood monocyte-derived dendritic cells express cell-surface mannose-specific lectins, we prepared various mannoses containing glycoconjugates with the aim of developing highly specific synthetic carriers of oligonucleotides and genes. Conjugates were prepared from oligosaccharides obtained by hydrazinolysis of Saccharomyces cerevisiae invertase glycopeptides. The reducing saccharides were converted into glycosynthons, i.e., into glyco-amino acids. Fluorescein derivatives were obtained by coupling the free carboxyl group of oligosaccharyl-pyroglutamate to the α-amino group of ε-fluoresceinyl-thiocarbamyl lysine methyl ester. It has been shown by others that glycosylated linear oligolysines containing up to six α-D-mannopyranosylphenylthiocarbamyl units have a high affinity for the human mannose receptor. In order to obtain fully biodegradable clusters and to improve both the specificity and the selectivity, disaccharides transformed into glycosynthons were coupled to pentalysine carriers (Lys5-Ala-Cys-NH2). Glycosylated pentalysyl cysteine conjugates were made fluorescent upon substitution of the cysteine thiol group with fluorescein iodoacetamide. As shown by flow cytofluorimetry, both the dimannoside clusters and yeast oligomannosides were very efficiently taken up by DC, conversely lactoside clusters were not.

Knowing that human blood monocyte-derived dendritic cells express cell-surface mannose-specific lectins, we prepared various mannoses containing glycoconjugates with the aim of developing highly specific synthetic carriers of oligonucleotides and genes. Conjugates were prepared from oligosaccharides obtained by hydrazinolysis of Saccharomyces cerevisiae invertase glycopeptides. The reducing saccharides were converted into glycosynthons, i.e., into glyco-amino acids. Fluorescein derivatives were obtained by coupling the free carboxyl group of oligosaccharyl-pyroglutamate to the α-amino group of ∊-fluoresceinyl-thiocarbamyl lysine methyl ester. It has been shown by others that glycosylated linear oligolysines containing up to six α-D-mannopyranosylphenylthiocarbamyl units have a high affinity for the human mannose receptor. In order to obtain fully biodegradable clusters and to improve both the specificity and the selectivity, disaccharides transformed into glycosynthons were coupled to pentalysine carriers (Lys5-Ala-Cys-NH2). Glycosylated pentalysyl cysteine conjugates were made fluorescent upon substitution of the cysteine thiol group with fluorescein iodoacetamide. As shown by flow cytofluorimetry, both the dimannoside clusters and yeast oligomannosides were very efficiently taken up by DC, conversely lactoside clusters were not.

The purpose of this study was to sequence the central part of the coding region of the Clostridium tyrobutyricumflagellin gene to improve the immunoenzymatic counting of cells after milk filtration. The coding region was amplified by PCR, and the amplified products were cloned. A DNA sequence analysis of positive clones gave us 1,131 nucleotides with a partial calculated flagellin molecular mass of 40,143 Da. The flagellar filament protein sequence exhibited high levels of homology to sequences of flagellin protein from other bacteria in both N‐ and C‐terminal parts, but little homology in the central domain. A PCR‐restriction fragment length polymorphism analysis of amplified C. tyrobutyricumflagellin gene products confirmed the variability of the central domain. The flagellin mRNA was determined to be 1.1 kb in size, which suggests a monocistronic mRNA. Furthermore, the deduced protein flagellin contains eleven potential N‐glycosylation sites and one sequence rich in serine, which could be modified by O‐glycosylation.

The monoclonal antibody 21E7‐B12 (IgG3) can be used in a direct method of Clostridium tyrobutyricumdetection based on an immunoenzymatic assay. Immunoelectron microscopy demonstrated that the 21E7‐B12 antibody recognized the surface‐exposed epitopes on the flagellar filaments of C. tyrobutyricum.After flagellar extraction, the purified flagellin showed an apparent molecular mass of 46 kDa with an isoelectric point of 3.6. Sugar staining, mild periodate oxidation and é‐elimination experiments showed that the flagellin was glycosylated and that the 21E7‐B12 epitope was located in the sugar moiety. Amino acid composition showed that the flagellar filament protein contained a high percentage of serine and threonine, while proline was absent. The first 23 residues of the N‐terminal were determined and sequence homology with other flagellins was found.

In order to count Clostridium tyrobutyricumspores in milk after membrane filtration, murine 21E7‐B12 monoclonal antibody was produced. Elution of the monoclonal antibody from this antigen, the flagellar filament protein, by carbohydrate ligands was used to study the epitope structure. A competitive elution of an anti‐dextran monoclonal antibody by carbohydrate ligands served as a control in order to validate the immunological tool applied to flagellin epitope study. The carbohydrate moiety of flagellin contained D‐glucose and N‐acetyl‐glucosamine in a molar ratio of 11:1 as determined by gas‐liquid chromatography and 2 low‐abundancy unidentified compounds. In ELISA, D‐glucose and N‐acetyl‐glucosamine did not dissociate the antibody‐flagellin complex contrary to maltose, maltotriose, maltotetraose and maltopentaose. The efficiency of elution increased from the dimer to the pentamer and became nil for maltohexaose and maltoheptaose. The fact that the hexamer and heptamer could not react with the 21E7‐B12 monoclonal antibody could be explained by a drastic conformational change. The overall stretched maltopentaose switch to a helical‐shaped maltoheptaose which could not fit the 21E7‐B12 monoclonal antibody antigen‐combining site. Thus, flagellin epitope may contain α(1→4) linked glucose residues plus either N‐actyl‐glucosamine or an unidentified compound that maintain it in an extended shape.